Combination of a lanthanum compound and bone enhancing agent for the treatment of bone diseases

ABSTRACT

The invention provides a method for enhancing bone formation, inhibiting osteoclastic differentiation and/or activating osteoblastic differentiation whereby to manage, treat or achieve prophylaxis of bone disease which comprises administering to a human or animal subject suffering from, or susceptible to bone disease a therapeutically or prophylactically effective amount of a lanthanum compound and a bone enhancing agent, such as vitamin D.

PRIORITY DATA

This application is a continuation-in-part of co-pending U.S.application Ser. No. 09/891,206, filed Jun. 26, 2001, which claimspriority to United Kingdom Application Serial No. 0015745.3, filed Jun.27, 2000. Each of the above applications are incorporated by referenceherein in their entirety.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to the treatment and prevention of bone diseases,to methods of enhancing bone formation and also to the treatment of bonefracture with a combination of a lanthanum compound and a bone enhancingagent, such as vitamin D.

BACKGROUND OF THE INVENTION

Throughout life, old bone is continuously removed by bone-resorbingosteoclasts and replaced with new bone which is formed by osteoblasts.This cycle is called the bone-remodeling cycle and is normally highlyregulated, i.e. the functioning of osteoclasts and osteoblasts is linkedsuch that in a steady state the same amount of bone is formed as isresorbed.

The bone-remodeling cycle occurs at particular areas on the surfaces ofbones. Osteoclasts which are formed from appropriate precursor cellswithin bones resorb portions of bone; new bone is then generated byosteoblastic activity. Osteoblasts synthesise the collagenous precursorsof bone matrix and also regulate its mineralization. The dynamicactivity of osteoblasts in the bone remodeling cycle to meet therequirements of skeletal growth and matrix and also regulate itsmaintenance and mechanical function is thought to be influenced byvarious factors, such as hormones, growth factors, physical activity andother stimuli. Osteoblasts are thought to have receptors for parathyroidhormone and estrogen. Ostoeclasts adhere to the surface of boneundergoing resorption and are thought to be activated by some form ofsignal from osteoblasts.

Irregularities in one or more stages of the bone-remodeling cycle (e.g.where the balance between bone formation and resorption is lost) canlead to bone remodeling disorders, or metabolic bone diseases. Examplesof such diseases are osteoporosis, Paget's disease and rickets. Some ofthese diseases are caused by over-activity of one half of thebone-remodeling cycle compared with the other, i.e. by osteoclasts orosteoblasts. In osteoporosis, for example, there is a relative increasein osteoclastic activity which may cause a reduction in bone density andmass. Osteoporosis is the most common of the metabolic bone diseases andmay be either a primary disease or may be secondary to another diseaseor other diseases.

Post-menopausal osteoporosis is currently the most common form ofosteoporosis. Senile osteoporosis afflicts elderly patients of eithersex and younger individuals occasionally suffer from osteoporosis.

Osteoporosis is characterised generally by a loss of bone density.Thinning and weakening of the bones leads to increased fracturing fromminimal trauma. The most prevalent fracturing in post-menopausalosteoporotics is of the wrist and spine. Senile osteoporosis, ischaracterised by a higher than average fracturing of the femur.

Whilst osteoporosis as a therapeutic target has been of, and continuesto, attract a great deal of interest, tight coupling between theosteoblastic and osteoclastic activities of the bone remodeling cyclemake the replacement of bone already lost an extremely difficultchallenge. Consequently, research into treatments for prevention orprophylaxis of osteoporosis (as opposed to replacement of already-lostbone) has yielded greater results to date.

Oestrogen deficiency has been considered to be a major cause ofpost-menopausal osteoporosis. Indeed steroids including oestrogen havebeen used as therapeutic agents (New Eng. J Med., 303, 1195 (1980)).However, recent studies have concluded that other causes must exist (J.Clin. Invest., 77, 1487 (1986)).

Other bone diseases can be caused by an irregularity in thebone-remodeling cycle whereby both increased bone resorption andincreased bone formation occur. Paget's disease is one such example.

Lanthanum has been of prominence previously in medicine on account ofits property of forming stable complexes with phosphate. Thisapplication has been evidenced in the treatment of hyperphosphataemia byapplication of lanthanum carbonate. U.S. Pat. No. 5,968,976 describesthe preparation and use in a pharmaceutical composition of certainhydrates of lanthanum carbonate for the treatment of hyperphosphataemia.

Fernandez-Gavarron et al. (Bone and Mineral, 283-291 (1988)) report onstudies into the incorporation of 140-lanthanum into bones teeth andhydroxyapatite in vitro. Whilst the depth of uptake varied from anestimated 5 to 15 μm (dependent on experimental conditions), theauthors' conclusion was that an exchange of lanthanum for calcium inhydroxyapatite may provide for increased resistance to acidic induceddissolution. Based on this suggested increased acid-resistance, theauthors suggest that lanthanum's clinical usefulness as an adjunct intreating diseases such as osteoporosis, root caries and alveolar boneresorption might be explored.

Vijai S.Shankar et al. (Biochemical and Biophysical ResearchCommunications, 907-912 (1992)) report that extracellular application ofLanthanum (III) induced a concentration-dependant elevation of cytosoliccalcium in osteoclasts. The authors suggested that the osteoclastcalcium receptor may be sensitive to activation and inactivation by thetrivalent cation lanthanum.

Bernd Zimmermann et al. (European Journal of Cell Biology, 114-121(1994)) report that lanthanum inhibited endochondral mineralization andreduced calcium accumulation in organoid cultures of limb bud mesodermalcells.

SUMMARY OF THE INVENTION

We have surprisingly found that lanthanum (III) compounds enhance boneformation and bone density and have beneficial effects on the activityand differentiation of bone cells.

Accordingly, the present invention relates to a method for enhancingbone formation in a mammal in need thereof comprising administering tothe mammal an effective amount of a lanthanum compound, preferablylanthanum (III). In accordance with an embodiment of the invention themammal is a human. The human may have a bone deficit or be at risk ofdeveloping a bone deficit. The invention also contemplates that thehuman has a bone remodeling disorder or is at risk of developing suchdisorder. Examples of bone remodeling disorders include osteoporosis,Paget's disease, osteoarthritis, rheumatoid arthritis, achondroplasia,osteochodrytis, hyperparathyroidism, osteogenesis imperfecta, congenitalhypophosphatasia, fribromatous lesions, fibrous displasia, multiplemyeloma, abnormal bone turnover, osteolytic bone disease and periodontaldisease.

In an embodiment the bone remodeling disorder is osteoporosis, includingprimary osteoporosis, secondary osteoporosis, post-menopausalosteoporosis, male osteoporosis and steroid induced osteoporosis.

Also provided is a method for enhancing bone formation in a mammalhaving a bone deficit which does not result from a bone remodelingdisorder. Such bone deficits may result, for example, from a bonefracture, bone trauma, or a condition associated with post-traumaticbone surgery, post-prosthetic joint surgery, post-plastic bone surgery,post-dental surgery, bone chemotherapy treatment or bone radiotherapytreatment.

In an embodiment of the methods of the invention the lanthanum (III)compound is lanthanum chloride, lanthanum carbonate, lanthanum salts,chelates or derivatives thereof, lanthanum resins or lanthanumabsorbants.

In a further embodiment of the methods of the invention, the effectiveamount of lanthanum (III) compound is from 0.01 mg/Kg/Day to 100mg/Kg/Day, preferably from 0.05 mg/Kg/Day to 50 mg/Kg/Day or from 0.1mg/Kg/Day to 10 mg/Kg/Day.

The present invention also provides a method for increasing bone densityin a mammal in need thereof comprising administering to said mammal aneffective amount of a lanthanum (III) compound. Also provided is amethod for stimulating osteoblast differentiation by contacting theosteoblasts with an effective amount of lanthanum (III) compound therebystimulating differentiation. Still further is provided a method forinhibiting osteoclast differentiation by contacting osteoclasts with aneffective amount of lanthanum (III) compound thereby inhibitingdifferentiation.

In a further embodiment, the invention provides a method for activatingthe bone formation activity of differentiated osteoblasts by contactingthe osteoblasts with an effective amount of lanthanum (III) compoundthereby stimulating bone formation. The invention also contemplates amethod for simultaneously stimulating osteoblast differentiation andinhibiting osteoclast differentiation in a mammal having a boneremodeling disorder, or being at risk of developing a bone remodelingdisorder, by administering to the mammal an effective amount oflanthanum (III) compound.

The invention also contemplates a method for enhancing bone formation ina mammal in need thereof by administering to the mammal an effectiveamount of a lanthanum (III) compound and at least one bone enhancingagent. Examples of suitable bone enhancing agents include a synthetichormone, a natural hormone, oestrogen, calcitonin, tamoxifen, abisphosphonate, a bisphosphonate analog, vitamin D, a vitamin D analog,a mineral supplement, a statin drug, a selective oestrogen receptormodulator and sodium fluoride.

The invention further contemplates the use of a lanthanum III compoundfor the preparation of a medicament for use in enhancing bone formationin a mammal in need thereof. In an embodiment the mammal is a humanhaving a bone remodeling disorder or being at risk of developing suchdisorder. In a further embodiment, the invention contemplates apharmaceutical composition for the treatment or prevention of a boneremodeling disorder comprising a lanthanum (III) compound and a boneenhancing agent.

The present inventors have also found that lanthanum compounds may beused to inhibit selectively osteoclast differentiation. At certain lowconcentrations osteoblast differentiation may be activated and increasedbone formation may result from the manifestation of either or both ofthese phenomena.

According to one aspect of the invention, there is thus provided amethod for inhibiting osteoclastic differentiation whereby to manage,treat or achieve prophylaxis of bone disease which comprisesadministering to a human or animal subject suffering from, orsusceptible to bone disease a therapeutically or prophylacticallyeffective amount of a lanthanum compound.

Viewed from a further aspect there is provided a method for activatingosteoblastic differentiation whereby to manage, treat or achieveprophylaxis of bone disease which comprises administering to a human oranimal subject suffering from, or susceptible to bone disease atherapeutically or prophylactically effective amount of a lanthanumcompound.

In this text, “susceptible to bone disease” is intended to embrace ahigher than average predisposition towards developing bone disease. Asan example, those susceptible towards osteoporosis includepost-menopausal women, elderly males (e.g. those over the age of 65) andthose being treated with drugs known to cause osteoporosis as aside-effect (e.g. steroid-induced osteoporosis).

According to a still further aspect of the invention there is providedthe use of a lanthanum compound for the preparation of a medicament foruse in any method of the invention.

According to a yet further aspect of the invention there is provided theuse of a lanthanum compound in any method of the invention.

According to a yet further aspect of the invention there is provided theuse of a lanthanum compound for the preparation of a pharmaceuticalcomposition for use in the diagnosis of bone disease or of bonefracture.

These and other aspects of the invention will become evident uponreference to the following detailed description and attached drawings.In addition reference is made herein to various publications which arehereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood by reference to the followingdrawings in which:

FIG. 1 is a bar graph showing the combined results of the effect of LAon bone resoption, where the bars represent the relative mediumCrossLaps amounts per osteoclast±SD at different lanthanumconcentrations;

FIG. 2 is a bar graph showing the combined results of the effect of LAon osteoclast differentiation, where the bars represent the relativeTRAP 5b activities±SD at different lanthanum concentrations;

FIG. 3 is a bar graph showing the combined results of the effect of LAon osteoblast differentiation, where the bars represent relativespecific activities of cellular alkaline phosphatase±SD at differentlanthanum concentrations; and

FIG. 4 is a bar graph showing the combined results of the effect of LAon bone formation activity of mature osteoblasts, where the barsrepresent the amount of calcium released (mmol/L) from the bone nodulesafter HC1 extraction. ±SD at different lanthanum concentrations.

DETAILED DESCRIPTION OF THE INVENTION

As hereinbefore mentioned, the present invention provides a method forenhancing bone formation in a mammal in need thereof comprisingadministering to said mammal an effective amount of a lanthanumcompound, preferably a lanthanum (III) compound, in combination with abone enhancing agent or agents.

Bone formation, or osteogenesis, refers to the creation of new bonemass. This includes the process whereby new bone structure grows or thedensity of existing bone is increased. Osteoblasts form bone byproducing extracellular organic matrix, or osteoid and then mineralizingthe matrix to form bone. The main mineral component of bone iscrystalline hydroxyapetite, which comprises much of the mass of normaladult bone.

As disclosed in parent application Ser. No. 09/891,206, the inventorshave surprisingly found that lanthanum compounds significantly enhancebone formation in vitro and in vivo. Enhanced bone formation in vitrowas observed when lanthanum (III) was added to cultures of matureosteoblasts in vitro at concentrations of from 100 to 15,000 ng/ml.Enhanced bone formation was quantitated by measuring the amount ofcalcium incorporated into bone nodules formed by the osteoblasts.

The present inventors have also found that lanthanum (III) enhanced boneformation in growing dogs. A dose of 2,000 mg/kg/day lanthanum enhancedbone formation and produced a significant increase in bone volume anddensity compared to control animals.

Lanthanum (III) compounds may be used in the methods of the invention toenhance bone formation in a range of mammals, including domesticanimals, such as pigs, cattle, horses, sheep and goats and alsoincluding pets and experimental mammals, such as dogs, cats and rodents.

In an embodiment of the invention the mammal is a human in need ofenhanced bone formation. In one aspect, the human in need has a bonedeficit, which means that they will have less bone than desirable orthat the bone will be less dense or strong than desired. A bone deficitmay be localized, such as that caused by a bone fracture or systemic,such as that caused by osteoporosis. Bone deficits may result from abone remodeling disorder whereby the balance between bone formation andbone resorption is shifted, resulting in a bone deficit. Examples ofsuch bone remodeling disorders include osteoporosis, Paget's disease,osteoarthritis, rheumatoid arthritis, achondroplasia, osteochodrytis,hyperparathyroidism, osteogenesis imperfecta, congenitalhypophosphatasia, fribromatous lesions, fibrous displasia, multiplemyeloma, abnormal bone turnover, osteolytic bone disease and periodontaldisease. Bone remodeling disorders include metabolic bone diseases whichare characterised by disturbances in the organic matrix, bonemineralization, bone remodeling, endocrine, nutritional and otherfactors which regulate skeletal and mineral homeostasis. Such disordersmay be hereditary or acquired and generally are systemic affecting theentire skeletal system.

Thus, in one aspect the human may have a bone remodeling disorder. Boneremodeling as used herein refers to the process whereby old bone isbeing removed and new bone is being formed by a continuous turnover ofbone matrix and mineral that involves bone resorption by osteoclasts andbone formation by osteoblasts.

Osteoporosis is a common bone remodeling disorder characterised by adecrease in bone density of normally mineralised bone, resulting inthinning and increased porosity of bone cortices and trabeculae. Theskeletal fragility caused by osteoporosis predisposes sufferers to bonepain and an increased incidence of fractures. Progressive bone loss inthis condition may result in a loss of up to 50% of the initial skeletalmass.

Primary osteoporosis includes idiopathic osteoporosis which occurs inchildren or young adults with normal gonadal function, Type Iosteoporosis, also described as post-menauposal osteoporosis, and TypeII osteoporosis, senile osteoporosis, occurs mainly in those personsolder than 70 years of age. Causes of secondary osteoporosis may beendocrine (e.g. glucocorticoid excess, hyperparathyroidism,hypoganodism), drug induced (e.g. corticosteroid, heparin, tobaco) orotherwise (e.g. chronic renal failure, hepatic disease and malabsorbtionsyndrome osteoporosis). The phrase “at risk of developing a bonedeficit”; as used herein, is intended to embrace mammals and humanshaving a higher than average predisposition towards developing a bonedeficit. As an example, those susceptible towards osteoporosis includepost-menopausal women, elderly males (e.g. those over the age of 65) andthose being treated with drugs known to cause osteoporosis as aside-effect (e.g. steroid-induced osteoporosis). Certain factors arewell known in the art which may be used to identify those at risk ofdeveloping a bone deficit due to bone remodeling disorders likeosteoporosis. Important factors include low bone mass, family history,life style, estrogen or androgen deficiency and negative calciumbalance. Postmenopausal women are particularly at risk of developingosteoporosis. Hereinafter, references to treatment of bone diseases areintended to include management and/or prophylaxis except where thecontext demands otherwise.

The methods of the invention may also be used to enhance bone formationin conditions where a bone deficit is caused by factors other than boneremodeling disorders. Such bone deficits include fractures, bone trauma,conditions associated with post-traumatic bone surgery, post-prostheticjoint surgery, post plastic bone surgery, post dental surgery, bonechemotherapy, post dental surgery and bone radiotherapy. Fracturesinclude all types of microscopic and macroscopic fractures. Examples offractures includes avulsion fracture, comminuted fracture, transversefracture, oblique fracture, spiral fracture, segmental fracture,displaced fracture, impacted fracture, greenstick fracture, torusfracture, fatigue fracture, intraarticular fracture (epiphysealfracture), closed fracture (simple fracture), open fracture (compoundfracture) and occult fracture.

As previously mentioned, a wide variety of bone diseases may be treatedin accordance with the present invention, for example all those bonediseases connected with the bone-remodeling cycle. Examples of suchdiseases include all forms of osteoporosis, osteomalacia, rickets andPaget's disease. Osteoporosis, especially of the post-menopausal, maleand steroid-induced types, is of particular note. In addition, lanthanumcompounds find use as antiresorption agents generally, as bone promotionagents and as anabolic bone agents. Such uses form another aspect of thepresent invention.

The present inventors have surprisingly found that lanthanum stimulatesosteoblast differentiation. Osteoblast differentiation was measured invitro cultures of bone marrow derived osteoprogenitor cells, which arecapable of proliferating and differentiating into mature osteoblasts,capable of forming mineralised bone nodules. Differentation was measuredby determining the specific activities of intracellular alkalinephosphatase. Low doses of lanthanum (100 ng/ml) were found to stimulateosteoblast differentiation.

The present inventors have also surprisingly found that lanthanuminhibits osteoclast differentiation in vitro, as measured by a decreasein TRAP (Tartrate-Resistant acid phosphatase) positive multinucleatecells in mouse bone marrow culture compared to control cultures. In manybone remodeling disorders, including osteoporosis, the bone deficit maybe attributed to excess bone resorption by differentiated osteoclasts.The methods and compositions of the invention may be employed to inhibitosteoclast differentiation, thus inhibiting bone resorption. Someinhibition of bone resorption was found in vitro.

Low doses of lanthanum have thus been found to both enhance boneformation and stimulate osteoblast differentiation and also to inhibitosteoclast differentiation and bone resorption.

A range of lanthanum compounds may be used in the methods andcompositions of the invention, preferably lanthanum (III) in a form thatis bioavailable. Preferred lanthanum compounds include, for example,lanthanum salts and derivatives thereof, lanthanum resins and lanthanumabsorbants. The lanthanum compound may, if desired, be in the form of achelate. Examples of suitable lanthanum salts include lanthanumcarbonate, lanthanum carbonate hydrate, lanthanum chloride.

An effective amount of lanthanum for use in the present invention is anamount of lanthanum (III)compound that will provide the desired benefitor therapeutic effect upon administration according to the prescribedregimen. Nonlimiting examples of an effective amount of lanthanum mayrange from about 0.01 mg/kg/day to about 100 mg/kg/day, preferably fromabout 0.05 mg/kg/day to about 50 mg/kg/day and more preferably fromabout 0.1 mg/kg/day to about 10 mg/kg/day.

The lanthanum compound orally administered to subjects in accordancewith this invention is suitably administered in unit dosages varyingfrom about 125 to about 1000 mg as elemental lanthanum. A typical dosagefor an adult can be, e.g., 375 mg-6000 mg daily. More preferably, thedosage is 375-3750 mg/day.

The dose may also be selected to provide an effective plasmaconcentration of lanthanum ion.

Examples of an effective plasma concentration of lanthanum ion may rangefrom about 0.1 ng/ml to about 1,000 ng/ml, preferably from about 1 ng/mlto about 500 ng/ml, more preferably from about 1 ng/ml to about 100ng/ml.

The dose may further be selected to provide an effective level oflanthanum in and around the bone surface.

Examples of effective amounts in and around the major bone surfaces mayrange from 0.1 μg/g to 500 μg/g, preferably from 0.5 μg/g to 100 μg/g,more preferably from 1 μg/g to 25 μg/g.

The term “lanthanum compound” is used herein to denote anypharmacologically acceptable lanthanum compound capable of ensuring thatthe lanthanum is bioavailable. Preferred compounds include, for example,lanthanum salts and derivatives thereof, lanthanum resins and lanthanumabsorbants. The lanthanum may if desired be in the form of a chelate.Hereinafter, the invention will be described with specific reference tocertain lanthanum salts and derivatives.

In accordance with the present invention, the lanthanum compound isadministered with a bone enhancing agent or agents. Bone enhancingagents are known in the art to increase bone formation, bone density orbone mineralisation, or to prevent bone resorption. Suitable boneenhancing agents include natural or synthetic hormones, such asestrogens, androgens, calcitonin, prostaglandins and parathormone;growth factors, such as platelet-derived growth factor, insulin-likegrowth factor, transforming growth factor, epidermal growth factor,connective tissue growth factor and fibroblast growth factor; vitamins,particularly vitamin D; minerals, such as calcium, aluminum, strontiumand fluoride; statin drugs, including pravastatin, fluvastatin,simvastatin, lovastatin and atorvastatin; agonists or antagonist ofreceptors on the surface of osteoblasts and osteoclasts, includingparathormone receptors, estrogen receptors and prostaglandin receptors;bisphosphonates and anabolic bone agents.

In one embodiment of the present invention, the lanthanum compound, incombination with vitamin D or an analog of vitamin D, is administered toa subject. Levels of 25-hydroxy vitamin D₂ are low at values less thanabout 16 ng/mL and replacement treatment aims for levels of greater thanor equal to about 16 ng/mL. Levels of 1,25-dihydroxy vitamin D₂ are lowat values less than about 22 pg/mL and replacement treatment aims forlevels of greater than about 22 pg/mL.

Examples of vitamin D sources which may be so administered concurrentlywith the lanthanum compound in this invention include 1,25dihydroxy-vitamin D, the active metabolite of vitamin D (calcitriol,rocalcitrol). Examples of suitable vitamin D analogs includedoxercalciferol (Hectorol®, available from Bone Care International,Middleton, Wis.), and paricalcitol (Zemplar®, available from AbbottLaboratories, Abbott Park, Ill.).

A pharmaceutical composition can contain up to about 1 mg of 25-hydroxyvitamin D₂ or 1,25-dihydroxy vitamin D₂. A pharmaceutical compositioncan also contain vitamin D₂ or vitamin D₃ in an amount up to 800 IU(where, e.g., vitamin D₂ is 850,000 IU/gram and vitamin D₃ is 100,000IU/gram).

The lanthanum compounds/bone enhancing agents of the invention may beadministered in the form of a pharmaceutical composition comprising theactive ingredients in admixture or association with a pharmaceuticallyacceptable carrier or diluent. The active ingredients may be formulatedinto a composition suitable for administration by any convenient route,e.g. orally (including sublingually), topically, parenterally (includingintravenous, intramuscular, intraperitoneal and subcutaneousadministration) and rectally, oral administration being preferred. Itshould be understood, however, that the invention embraces all forms ofadministration which make the lanthanum compound and bone enhancingagent(s) systemically or locally available. Orally administrablecompositions may, if desired, contain one or more physiologicallycompatible carriers and/or excipients and may be solid or liquid. Thecompositions may take any convenient form including, for example,tablets, coated tablets, capsules, lozenges, aqueous or oilysuspensions, solutions, emulsions, syrups, elixirs and dry productssuitable for reconstitution with water or another suitable liquidvehicle before use. The compositions may advantageously be prepared inunit dosage form. Tablets and capsules according to the invention may,if desired, contain conventional ingredients such as binding agents, forexample syrup, acacia, gelatin, sorbitol, tragacanth orpolyvinyl-pyrollidone; fillers, for example lactose, sugar, maizestarch, calcium phosphate, sorbitol or glycine; lubricants, for examplemagnesium stearate, talc, polyethylene glycol or silica; disintegrants,for example potato starch; or acceptable wetting agents such as sodiumlauryl sulphate. Tablets may be coated according to methods well knownin the art.

Furthermore, dextrates can be an ingredient in a composition of theinstant invention. The term “dextrates” as used herein refers to apurified mixture of saccharides that is mostly dextrose (e.g., not lessthan about 93.0% and not more than about 99.0%, calculated on the driedbasis) and that results from a controlled enzymatic hydrolysis ofstarch. Dextrates can be either anhydrous or hydrated. “Dextrates” canrefer to dextrates as defined its official monograph found in NationalFormulary 21 (printed by Webcom Limited in Toronoto, Cnada; 2003).Dextrates are available from JRS Pharma (Patterson, N.Y.) as Emdex®.

Liquid compositions may contain conventional additives such assuspending agents, for example sorbitol syrup, methyl cellulose,glucose/sugar syrup, gelatin, hydroxymethylcellulose,carboxymethylcellulose, aluminium stearate gel or hydrogenated ediblefats; emulsifying agents, for example lecithin, sorbitan monooleate oracacia; non-aqueous vehicles, which may include edible oils, for examplevegetable oils such as arachis oil, almond oil, fractionated coconutoil, fish-liver oils, oily esters such as polysorbate 80, propyleneglycol, or ethyl alcohol; and preservatives, for example methyl orpropyl p-hydroxybenzoates or sorbic acid. Liquid compositions mayconveniently be encapsulated in, for example, gelatin to give a productin dosage unit form.

Formulations for oral delivery may be formulated in a delayed releaseformulation such that the lanthanum is delivered to the large intestine.This will lessen the interaction of lanthanum with dietary phosphatewhich results in the precipitation of lanthanum phosphate, which ispoorly absorbed by the gut. Delayed release formulations are well knownin the art and include for example, delayed release capsules or timepills, osmotic delivery capsules etc.

Compositions for parenteral administration may be formulated using aninjectable liquid carrier such as sterile pyrogen-free water, sterileperoxide-free ethyl oleate, dehydrated alcohol or propylene glycol or adehydrated alcohol/propylene glycol mixture, and may be injectedintravenously, intraperitoneally, subcutaneously or intramuscularly.

Compositions for rectal administration may be formulated using aconventional suppository base such as cocoa butter or another glyceride.

Compositions for topical administration include ointments, creams, gels,lotions, shampoos, paints, powders (including spray powders), pessaries,tampons, sprays, dips, aerosols, pour-ons and drops. The activeingredient may, for example, be formulated in a hydrophilic orhydrophobic base as appropriate.

It may be advantageous to incorporate an antioxidant, for exampleascorbic acid, butylated hydroxyanisole or hydroquinone in thecompositions of the invention to enhance their storage life.

Administration in this invention may consist of one or more cycles;during these cycles one or more periods of osteoclastic and osteoblasticactivity will occur, as well as one or more periods when there isneither osteoclastic nor osteoblastic activity.

Alternatively, administration may be conducted in an uninterruptedregimen; such a regimen may be a long term regimen, e.g. a permanentregimen.

It will be understood that the dosages of compositions and the durationof administration according to the invention will vary depending on therequirements of the particular subject. The precise dosage regime willbe determined by the attending physician or veterinary surgeon who will,inter alia, consider factors such as body weight, age and symptoms (ifany). The compositions may if desired incorporate one or more furtheractive ingredients.

During the dosing regimen, administration may be effected once or moretimes per day, for example once, twice, three or four times per day.

FIGS. 1 to 4 show the effect of the lanthanum (III) ion on boneresorption, osteoclast differentiation, osteoblast differentiation andbone formation respectively.

The following non-limiting examples describing the effect of a lanthanum(III) ion-containing solution in vitro bone culture assays and in vivostudy are illustrative of the present invention.

EXAMPLE 1 In Vitro Bone Resorption Assay

Test Substance

The test substance was lanthanum carbonate tetrahydrate (hereinafterlanthanum carbonate). 1 mg of lanthanum is equivalent to 1.9077 mg oflanthanum carbonate. Lanthanum carbonate was dissolved in 2M HCl to givea concentration of 28.6 mg/ml (i.e 15 mg/ml of lanthanum).

Aliquots of this stock solution were diluted with 2M HCl to result insolutions of varying concentrations, so that addition of one microliterof these solutions into the culture medium gave the final testconcentrations of 100, 500, 1000, 5000 and 15000 ng/ml of lanthanum inculture medium. These solutions/concentrations are hereinafter referredto as LA100, LA500, LA1000, LA5000 and LA15000.

Control Substances

We used control groups in each assay to show that the assays werecapable of detecting the effect of inhibition (bone resorption assay andosteoclast differentiation assay) or activation (osteoblastdifferentiation and bone formation). The control substances used were:

-   -   Bafilomycin Al (in bone resorption assay)    -   17β-estradiol (in osteoblast differentiation assay and    -   bone formation assay)

In the osteoclast differentiation assay, the control group did notcontain vitamin D.

The method of osteoclast culture on bone slices was originally describedby Boyde et al. (1984) and by Chambers et al. (1984). For cell culture,we used a method slightly modified from the original methods (Lakkakorpiet al. 1989, Lakkakorpi and Väänännen, 1991). The rate of boneresorption in the cultures was originally determined by counting thenumber of resorption pits on each bone or dentine slice using amicroscope with phase contrast objectives (Sundquist et al. 1990).Later, the pits were visualized using Wheat Germ Agglutinin lectin thatspecifically binds to the resorbed area in bone (Selander et al. 1994),making it possible to quantify the total resorbed area using amicroscope and computer-assisted image analysis system (Laitala andVäänänen 1994, Hentunen et al. 1995). We used a commercially availablemethod (CrossLaps for cultures, Osteometer Biotech, Herlev, Denmark) todetect the amount of collagen cross-links released into the culturemedium as an index of the bone resorption rate (Bagger et al., 1999).

The study protocol uses a method where osteoclasts are cultured on boneslices and allowed to resorb bone. The system is ideal for determiningthe effect of drug candidates on the bone resorbing activity ofosteoclasts. Drug candidates are added into the cell cultures at thebeginning of the culture period, and the osteoclasts allowed to resorbbone for 3 days. The amount of bone resorbed during the culture periodis determined and compared to the amount of bone resorbed in controlcultures (those cultured in the absence of drug candidates). If the drugcandidate inhibits the function of osteoclasts, the amount of boneresorbed in these cultures is significantly lower than in the controlcultures.

Procedure:

Transverse 0.1 mm thick slices of cortical bone were cut from thediaphysis of fresh bovine femurs (Atria Slaughterhouse, Oulu, Finland)using a low-speed diamond saw, cleaned by ultrasonication in multiplechanges of sterile distilled water, and stored at 4° C. before use. Longbones were removed from 1-day-old rat pups killed by decapitation. Thebones were dissected free of adherent soft tissues, and the endostealsurfaces were curetted with a scalpel blade into the osteoclast culturemedium (Dulbecco s Modified Eagle s Medium (DMIEM), (Gibco BRL, Paisley,UK)) supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin(Penicillin/Streptomycin solution, Gibco BRL, Paisley, UK), 20 mM HEPESbuffer (Gibco BRL, Paisley, UK) and 10% heat-inactivated fetal calfserum, pH 6.9 (Gibco BRL, Paisley, UK). The resulting suspension ofdispersed cells and bone fragments was agitated using a plastic pipette.Larger fragments were allowed to sediment for a few seconds and thesupernatant was seeded onto the bone slices pre-wetted in the medium.After a settling period of 30 minutes at 37° C., the bone slices werewashed by dipping in fresh medium, and then transferred to wells in24-well culture dishes containing osteoclast culture medium. The boneslices were incubated in a humidified atmosphere of 95% air and 5%carbon dioxide at 37° C. for 72 hours.

After the culture period, the amount of bone resorption was determinedby measuring the amount of collagen cross-links released into theculture medium using a commercial kit (CrossLaps for cultures,Osteometer Biotech) according to the manufacturer's instructions. Thenumber of osteoclasts in each culture was determined by microscopiccounting of the amount of TRAP-positive multinuclear cells, and theresults are given as the number of collagen cross-links released per oneosteoclast.

In this study, the effect of the lanthanum (III) ion on the boneresorbing activity of osteoclasts was tested.

The following sample groups were included:

-   -   Baseline (including vehicle)    -   Control (Baseline+10 nM bafilomycin Al)    -   Baseline+100 ng/ml lanthanum    -   Baseline+500 ng/ml lanthanum    -   Baseline+1000 ng/ml lanthanum    -   Baseline+5000 ng/ml lanthanum    -   Baseline+15000 ng/ml lanthanum

Six replicates were included in each group, and the test was performedtwice. Bafilomycin Al, a highly potent inhibitor of osteoclast V-ATPaseproton pump, was used as a control to show the ability of the testsystem to detect inhibition of bone resorption.

Tables of Results:

In the bone resorption assay, the amount of medium CrossLaps (nM)released into the culture medium was determined and the number ofosteoclasts in the corresponding cultures calculated. The mediumCrossLaps amounts were divided with the osteoclast numbers in thecorresponding cultures, and the results are given on Table 1 as relativemedium CrossLaps amounts per osteoclasts. The relative values wereobtained by dividing each individual value with the mean value of thebaseline group. TABLE 1 Relative medium CrossLaps amounts per osteoclastin the first bone resorption assay Group 1 2 3 4 5 6 Mean ± SD Baseline0.98 0.82 1.01 1.65 0.74 0.81 1.00 ± 0.34 Control 0.00 0.00 0.00 0.190.27 0.14 0.10 ± 0.11(***) LA 100 0.57 0.56 1.13 0.78 0.71 0.71 0.74 ±0.21 LA 500 1.04 0.58 1.38 0.75 0.88 0.63 0.88 ± 0.30 LA 1000 1.14 1.090.89 1.76 1.07 1.11 1.18 ± 0.30 LA 5000 1.39 0.78 2.70 1.18 0.76 1.211.34 ± 0.71 LA 15000 0.57 0.58 0.57 0.96 2.53 1.11 1.05 ± 0.76

TABLE 2 Relative medium CrossLaps amounts per osteoclasts in the secondbone resorption assay Group 1 2 3 4 5 6 Mean ± SD Baseline 0.75 1.330.88 1.98 0.53 0.53 1.00 + 0.56 Control 0.00 0.00 0.00 0.00 0.00 0.000.00 ± 0.00(***) LA 100 0.38 0.75 0.78 0.94 0.67 0.96 0.74 ± 0.21 LA 5000.50 2.14 0.50 1.03 0.47 0.63 0.88 ± 0.65 LA 1000 0.70 0.59 1.69 1.401.68 0.73 1.13 ± 0.51 LA 5000 0.48 1.18 0.77 0.98 1.99 1.81 1.20 + 0.59LA 15000 0.29 1.08 0.62 0.87 0.47 0.45 0.63 + 0.29

All data shown on tables 1 and 2 were combined and analyzed. Thecombined results are shown on table 3 and FIG. 1. TABLE 3 Combinedresults of the effect of LA 100-LA 15000 on bone resorption Group numberMean ± SD Baseline 12 1.00 ± 0.44 Control 12 0.00 ± 0.00(***) LA 100 120.74 ± 0.20 LA 500 12 0.88 ± 0.48 LA 1000 12 1.15 ± 0.40 LA 5000 12 1.27± 0.63 LA 15000 12 0.84 ± 0.59Results

In the bone resorption assay, there was no significant effect of thelanthanum (III) ion on either the amount of CrossLaps released into theculture medium or on the osteoclast number. The control substance,bafilomycin Al, completely inhibited bone resorption. As shown on table3 and FIG. 4, the lanthanum (III) ion has no statistically significanteffects on the bone resorbing activity of individual mature osteoclastsat any of the concentrations tested. However, the dose-dependentinhibition of bone resorption with the lower concentrations (LA 100 andLA 500) should be noticed. The slight decrease seen with LA 15000 may bedue to slight toxic effects of this high concentration.

REFERENCE

Bagger Y Z, Foged N T, Andersen L, Lou H, Qvist P (1999) CrossLaps forculture: An improved enzyme-linked immunosorbent assay (ELISA) formeasuring bone resorption in vitro. J Bone Miner Res 14, Suppl. 1, S370.

Boyde A, Ali N N, Jones Si (1984) Resorption of dentine by isolatedosteoclasts in vitro. Br Dcnt J 156: 216-220.

Chambers T J, Revell P A, Fuller K, Athanasou N A (1984) Resorption ofbone by isolated rabbit osteociasts. J Cell Sci 66: 383-399.

Hentunen T A, Lakkakorpi P T, Tuukkanen J, Lehenkari P P, Sampath T K,Väänänen B K (1995) Effects of recombinant human osteogenic protein-1 onthe differentiation of osteoclast-like cells and bone resorption.Biochem Biophys Res Commun 209: 433-443.

Laitala T, Väänännen H K (1994) Inhibition of bone resorption in vitroby antisense RNA and DNA molecules targeted against carbonic anhydraseII or two subunits of vacuolar H+-ATPase. J Clin Invest 93: 2311-2318.

Lakkakorpi F, Tuukkanan I, Hentunen T, Jarvelin K, Väänänen H K (1989)Organization of osteoclast microfilaments during the attachment to bonesurface in vitro. I Bone Miner Res 4: 817-825.

Lakkakorpi P T, Väänänen H K (1991) Kinetics of the osteoclastcytoskeleton during the resorption cycle in vitro. J Bone Miner Res 6:817.826.

Selander K, Lehenkari P, Väänänen H K(1994) The effects ofbisphosphonates on the resorption cycle of isolated osteoclasts. CalcifTissue Int 55: 368-375.

Sundquist K, Lakkakorpi P, Wallmark B, Väänänen H K (1990) Inhibition ofosteoclast proton transport by bafilomycin A₁ abolishes bone resorption.Biochem Biophys Res Commun 168: 309-313.

EXAMPLE 2 In Vitro Osteoclast Differentiation Assay

A method known as mouse bone marrow culture system is the one mostwidely used to study osteoclast differentiation. Originally, this methodwas developed by Takahashi et al. (1988a). Osteoclast precursors inmouse bone marrow can be induced to form multinucleated osteoclast-likecells (MNC) in the presence of either an active metabolite of vitamin D₃(1,25(OH)₂D₃) or parathyroid hormone (PTH). MNC formed in mouse bonemarrow cultures have been demonstrated to possess several featurescharacteristic of osteoclasts. They form pits on bone or dentine slices(Takahashi et al. 1988a, Hattersley and Chambers 1989, Shinar et al.1990); they express high levels of tartrate-resistant acid phosphatase(TRAP) and calcitonin receptors (Takahashi et al. 1988b, Shinar et al.1990); and they respond to calcitonin (Takahashi et al. 1988a) andprostaglandin E₂ (Collins and Chambers 1992). Thus, the method is anideal one with which to study both stimulators and inhibitors ofosteoclast differentiation.

In the original culture system, the osteoclast formation was determinedafter an 8-day culture. In bone marrow, both non-adherent osteoclastprecursors and stromal cells are present, the latter of which are neededto support osteoclast formation. The number of osteoclasts formed isgenerally determined by counting the number of TRAP-positive MNCcontaining at least three nuclei (Takahashi et al. 1988a). In thenegative control, where 1,25(OH)₂D₃ is not added, TRAP-positive MNC arenot formed.

We have modified the original assay so that we culture 1×10⁶ mousemarrow cells/ml for 6 days. With this modification, the number ofTRAP-positive MNC/culture has been shown to be approximately 150 (Choiet al. 1998, Hentunen et al. 1998). Instead of counting of the number ofdifferentiated osteoclasts formed, we measured the amount of TRAPliberated from osteoclasts into the culture medium using a fast, simpleTRAP immunoassay (Halleen et al. 1999) presentation in the AnnualMeeting of the American Society for Bone and Mineral Research, Sep.30-Oct. 4, 1999, in St. Louis, Mo., USA. Our results show that theamount of TRAP released into the culture medium correlates significantly(r=0.94, p<0.0001, n=120) with the amount of osteoclasts formed.

Procedure:

8-10-week old mice were killed with CO₂. Tibia and femora were dissectedfree from adhering soft tissues. The bone ends were cut off with ascalpel and the marrow was flushed with α-Minimal Essential Medium(α-MEM, Gibco BRL, Paisley, UK) supplemented with 100 IU/ml penicillinand 100 μg/ml streptomycin. A 10 ml syringe with a 27 gauge needle wasused for flushing. Cells were centrifuged at 600×G for 10 minutes andthe cell pellet was resuspended in α-MEM containing 10% fetal calfserum. Cells were allowed to attach to plastic for 2 h at 37° C. in a 5%CO₂ incubator to allow removal of monocytes and macrophages. Nonadherentcells were duly removed, and the attached bone marrow cells werecultured in 24-well plates (1×10⁶ cells/well=1 ml) for 6 days. Half ofthe media were changed at day 3 and the treatments replaced. At the endof the culture, the plates were fixed with 2% paraformaldehyde in PBSfor 20 minutes. Osteoclast formation was determined by measuring TRAPactivity from the culture media using the novel TRAP immunoassay (videinfra), where we use a polyclonal TRAP antiserum prepared in rabbitsagainst purified human bone TRAP. The TRAP antibody was bound toanti-rabbit IgG coated microtiter wells (Gibco BRL, Paisley, UK), andmedium TRAP was then bound to the antibody. The activity of bound TRAPwas measured in sodium acetate buffer using pNPP as substrate.

In this study, the effect of the lanthanum (III) ion on osteoclastdifferentiation in the presence of 1,25-dihydroxyvitamin D3 was tested.The following sample groups were included:

-   -   Baseline (including vehicle)    -   Control (Baseline without 1,25-dihydroxyvitamin D3)    -   Baseline+100 ng/ml lanthanum    -   Baseline+500 ng/ml lanthanum    -   Baseline+1000 ng/ml lanthanum    -   Baseline+5000 ng/ml lanthanum    -   Baseline+15000 ng/ml lanthanum

Six replicates were included in each group, and the test was performedtwice. Baseline without 1,25-dihydroxyvitamin D3 was used as a controlto show the test system allows inhibition of osteoclast differentiationto be detected. As the results of LA100 did not give statistically thesame result (significantly different or not compared with the baseline)in both of the two tests, we performed the test with LA100 oneadditional time.

Tables of Results:

In the osteoclast differentiation assay, the amount of TRAP 5b activityreleased into the culture medium was determined as an index ofosteoclast number. The results are shown as relative TRAP 5b activitiesobtained by dividing each individual TRAP 5b activity with the mean TRAP5b activity of the baseline group. TABLE 4 Relative TRAP 5b activitiesin the first osteoclast differentiation assay Group 1 2 3 4 5 6 Mean ±SD Baseline 1.32 0.72 0.43 0.45 1.89 1.18 1.00 + 0.57 Control 0.16 0.170.18 0.11 0.11 0.20 0.16 ± 0.04(**) LA 100 0.81 0.96 0.43 1.39 0.98 0.650.87 + 0.33 LA 500 0.73 0.55 0.48 0.87 0.58 1.05 0.71 ± 0.22 LA 10000.58 0.82 0.35 0.40 0.98 0.45 0.60 + 0.25 LA 5000 0.44 0.40 0.41 0.360.51 0.52 0.44 ± 0.06(*) LA 15000 0.14 0.26 0.21 0.34 0.31 0.88 0.36 ±0.27(*)

TABLE 5 Relative TRAP 5b activities in the second osteoclastdifferentiation assay Group 1 2 3 4 5 6 Mean ± SD Baseline 1.27 1.370.98 0.92 0.74 0.71 1.00 + 0.27 Control 0.17 0.34 0.14 0.10 0.11 0.060.15 ± 0.10(***) LA 100 0.64 0.66 0.62 0.36 0.33 0.62 0.54 ± 0.15(**) LA500 1.16 1.30 0.85 1.33 0.76 1.01 1.07 ± 0.24 LA 1000 0.70 0.78 0.340.65 0.69 1.00 0.69 + 0.21 LA 5000 0.94 0.46 0.21 0.72 0.68 0.33 0.56 ±0.27(*) LA 15000 0.22 0.31 0.35 0.25 0.15 0.20 0.25 ± 0.07(**)

The assay with LA 100 was repeated one more time, because the resultswere significantly different from baseline in the second assay, and notsignificantly different in the first assay. TABLE 6 Relative TRAP 5bactivities in the third osteoclast differentiation assay with LA 100.Group 1 2 3 4 5 6 Mean ± SD Baseline 1.25 1.20 0.76 0.93 1.07 0.811.00 + 0.20 Control 0.08 0.07 0.20 0.10 0.25 0.13 0.14 ± 0.07(***) LA100 0.71 0.96 0.42 0.47 0.87 0.69 0.69 ± 0.21(*)

All data shown on tables 4-6 were combined and analyzed. The combinedresults are shown on table 7 and FIG. 2. TABLE 7 Combined results of theeffect of LA 100-LA 15000 on osteoclast differentiation Group numberMean ± SD Baseline 18 1.00 ± 0.36 Control 18 0.15 ± 0.07(***) LA 100 180.70 ± 0.27(**) LA 500 12 0.89 ± 0.29 LA 1000 12 0.65 ± 0.23(**) LA 500012 0.50 ± 0.20(***) LA 15000 12 0.30 ± 0.19(***)Results:

In the osteoclast differentiation assay, a clear dose-dependentinhibition was observed with LA 500-LA 15000 that was statisticallysignificant from LA 1000 to LA 15000. A statistically significantinhibition was also observed with LA 100. In the control group wherevitamin D was omitted, osteoclast differentiation was significantlylower than in the baseline group.

REFERENCES

Halleen N, Alatalo S, Hentunen T A, Väänänen H K (1999) A novel TRAP 5bimmunoassay for osteoclast cultures. J Bone Miner Res 14, Suppl. 1,S244.

Choi S J, Devlin R D. Menaa C, Chung H, Roodman G D, Reddy S V (1998)Cloning and identification of human Sca as a novel inhibitor ofosteoclast formation and bone resorption. J Clin Invest 102: 1360-1368.

Collins D A, Chambers T J (1992) Prostaglandin E₂ promotes osteoclastformation in murine hematopoietic cultures through an action onhematopotetic cells. J Bone Miner Res 7: 555-561.

Hattersley G, Chambers T J (1989) Generation of osteoclastic function inmouse bone marrow cultures: multinuclearity and tartrate-resistant acidphosphatase are unreliable markers for ostcoclastic differentiation.Endocrinology 124: 1689-1696.

Hentunen T A, Reddy S V. Boyce B F, Dovlin R, Park H-R, Chimg H,Selander K S, Dallas M, Kurihara N, Galson O L, Goldring S R, Koop, B AWindle J J, Roodman G D (1998) Immortalization of osteoclast precursorsby targeting bcl-X_(L), and simian virus 40 large T antigen to theosteoclast lineage in transgenic mice. J Clin Invest 102: 88-97.

Shinar D M, Sato M, Rodan G A (1990) The effect of hemopoietic growthfactors on the generation of osteoclast-like cells in mouse bone marrowcultures. Endocrinology 126: 1728-1735.

Takahashi N, Yamana H, Yoshiki S, Roodman G D, Mundy G R, Jones S J,Boyde A, Suda T (1988a) Osteoblast-Like cell formation and itsregulation by osteotropic hormones in mouse bone marrow cultures.Endocrinology 122:1373-1382.

Takahashi N. Akatsu T, Sasaki T, Nicholson G C, Moseley J M, Martin T J,Suda T (1988b) Induction of calcitonin receptors by 1,25-dihydroxyvitamin D₃ in osteoblast-like multinucleated cells formedfrom mouse bone marrow cells. Endocrinology 123: 1504-1510.

EXAMPLE 3 In Vitro Osteoblast Differentiation Assay

Osteoblasts are bone-forming cells which arise from mesenchymal stemcells. During the development of osteoblasts, three distinct periodshave been identified and defined: 1) cell proliferation and secretion ofextracellular matrix (ECM); 2) ECM maturation; and 3) ECMmineralization. During these periods, a sequential expression ofosteoblast phenotype markers has been characterized. Alkalinephosphatase is associated with the bone cell phenotype and is activelyexpressed during the maturation of the osteoblast. With the onset ofmineralization, large amounts of calcium are deposited into the matureorganic matrix to form bone-like nodules. By following these markers, weare able to study all the stages of osteoblast differentiation in thisculture system.

Several methods have been devised to study osteoblasts. The first ofthese involves isolation of cells from calvaria with the osteoblasticphenotype. However, these cells only represent the mature stage ofosteoblasts, because only a small fraction of the calvarial cells areosteoblast precursors (Bellows and Aubin 1989, Bellows et al. 1994).Osteoblastic cell lines are convenient in use, but they may not behaveas primary osteoblasts (Mundy 1995). It is conceivable that osteoblastprecursors are present in bone marrow (Friedenstein 1976, Owen 1988),and bone marrow stromal cells have long been recognized as the source ofosteoprogenitor cells.

We have established a culture model in which mouse bone marrow derivedosteoprogenitor cells first proliferate and then differentiate toosteoblasts capable of forming mineralized bone nodules (Qu et al. 1998,Qu et al. 1999). We confirmed this by following the expression ofseveral markers of the osteoblastic phenotype and by studying themorphology of cultures at light and electron microscopic level.Synthesis of fibrillar extracellular matrix with late deposition ofcalcium confirmed the differentiation and maturation of osteoblasts.Thus, this culture system fulfills requirements of an in vitro modeluseful for studying differentiation of osteoprogenitor cells into bonesynthesizing osteoblasts.

Procedure:

Bone marrow cells were obtained from the femurs of 10-week old femaleNMRI mice. Animals were killed by cervical dislocation. Both femora wereremoved and the soft tissues were detached aseptically. Metaphyses fromboth ends were cut off and bone marrow cells were collected by flushingthe diaphysis with culture medium: phenol red-free-a-modified essentialmedium (α-MEM (Gibco BRL, Paisley, UK)). A suspension of bone marrowcells was obtained by repeated aspiration of the cell preparationthrough a 22 gauge needle, and nucleated cells were counted with ahemocytometer. Cells were plated at 10⁶ cells/cm² in T-75 tissue cultureflasks in phenol red-free-α-MEM supplemented with 10% FCS, 10⁻⁸ Mdexamethasone, 50 μg/ml ascorbic acid, 10−2 M sodium β-glycerophosphate,100 IU/ml penicillin and 100 μg/ml streptomycin. The cells were culturedfor 6 days and half of the media replaced after 3 days. On day 6,subcultures were prepared. Cells were washed with warm PBS and adherentcells were detached using trypsin-EDTA. Trypsinized cells were passedthrough a syringe with a 22 gauge needle to make a single-cellsuspension, counted and plated in 24-well plates at a density of 5×10³cells/ml. These osteoprogenitor cells were stimulated to differentiatetowards mature osteoblasts by culturing them in the presence of 10⁻¹⁰ Mestrogen (17β-estradiol) for 8 days. The test substances were added atthe beginning of the secondary culture without estrogen, and every timewhen the medium was changed.

The number of osteoblasts formed was determined by measuring cellularalkaline phosphatase (ALP) activity in the culture. Cells were disruptedby washing the cell layers twice with PBS, extracting into 200 μl 0.1%Triton X-100 buffer at pH 7.6 (Sigma, St. Louis, Mo., USA), andovernight freezing. ALP activity was determined calorimetrically usingp-nitrophenylphosphate as substrate at pH 9.7 and determining theoptical density at 405 nm. In parallel, protein contents of the wellswere determined by the BIO-RAD protein assay, and the specific ALPactivity is expressed as units/mg protein.

In this study, the effect of the lanthanum (III) ion on osteoblastdifferentiation was tested. The following sample groups were included:

-   -   Baseline (+vehicle)    -   Control (Baseline+10-10⁻¹⁰ M 17 -estradiol)    -   Baseline+100 ng/ml lanthanum    -   Baseline+500 ng/ml lanthanum    -   Baseline+1000 ng/ml lanthanum    -   Baseline+5000 ng/ml lanthanum    -   Baseline+15000 ng/ml lanthanum        Tables of Results:

Osteoblast differentiation was determined by measuring cellular alkalinephosphatase (ALP) activities and total protein amounts from celllysates. The ALP activities were divided with the corresponding proteinamounts to obtain specific activities of ALP. The results are shown asrelative specific activities obtained by dividing each individual valuewith the mean value of the baseline group. TABLE 8 Relative specificactivities of intracellular alkaline phosphatase in the preliminaryosteoblast differentiation assay Group 1 2 3 4 Mean ± SD Baseline 0.941.10 0.94 1.02 1.00 + 0.07 Control 1.10 1.32 1.31 1.29 1.26 ± 0.10(**)LA 100 0.98 1.29 1.19 1.12 1.15 + 0.13 LA 500 0.96 0.98 0.99 1.11 1.01 +0.07 LA 1000 0.69 1.13 0.92 1.01 0.94 ± 0.19 LA 5000 0.42 0.46 0.50 0.480.47 ± 0.03(***) LA 15000 0.51 0.49 0.47 0.54 0.50 ± 0.03(***)

TABLE 9 Relative specific activities of intracellular alkalinephosphatase in the first osteoblast differentiation assay Group 1 2 3 45 6 7 8 Mean ± SD Baseline 0.97 0.94 1.12 0.98 0.97 1.06 0.99 0.96 1.00± 0.06 Control 1.01 1.20 1.04 1.13 1.19 1.06 1.03 1.14 1.10 ± 0.08(**)LA 100 1.25 0.98 1.31 0.77 0.95 1.04 1.13 0.98 1.05 + 0.17 LA 500 0.831.03 1.02 0.98 0.95 0.96 0.82 0.62 0.90 + 0.14 LA 1000 1.01 1.12 1.060.76 1.01 0.78 0.93 0.81 0.94 + 0.14 LA 5000 0.54 0.48 0.47 0.63 0.540.59 0.44 0.55 0.53 ± 0.06(***) LA 15000 0.40 0.42 0.53 0.36 0.39 0.350.30 0.43 0.40 ± 0.07(***)

TABLE 10 lative specific activities of intracellular alkalinephosphatase in the second osteoblast differentiation assay Group 1 2 3 45 6 Mean ± SD Baseline 0.99 0.83 1.25 1.01 0.88 1.04 1.00 ± 0.15 Control1.00 1.18 1.53 1.52 1.03 1.38 1.27 ± 0.24(*) LA 100 0.91 0.94 1.34 1.201.00 1.43 1.14 + 0.22 LA 500 0.88 0.89 1.10 1.09 0.75 0.90 0.93 ± 0.14LA 1000 0.73 0.71 1.19 0.81 0.72 1.09 0.88 + 0.21 LA 5000 0.31 0.51 0.510.49 0.28 0.40 0.41 ± 0.10(***) LA 15000 0.27 0.13 0.33 0.32 0.29 0.310.28 ± 0.07(***)

All data shown on tables 8-10 were combined and analyzed. The combinedresults are shown on table 11 and FIG. 3. TABLE 11 Combined results ofthe effect of LA 100-LA 15000 on osteoblast differentiation Group numberMean ± SD Baseline 18 1.00 ± 0.09 Control 18 1.19 ± 0.17(***) LA 100 181.10 ± 0.18(*) LA 500 18 0.94 ± 0.13 LA 1000 18 0.92 ± 0.17 LA 5000 180.48 ± 0.09(***) LA 15000 18 0.38 ± 0.11(***)Results:

The lanthanum (III) ion showed a clear dose-dependent response in theosteoblast differentiation assay. The highest test concentrations (LA5000 and LA 15000) inhibited, and the lowest test concentration (LA 100)activated osteoblast differentiation significantly. No significantresponse was observed with LA 500 and LA 1000. The control substance,17β-estradiol, activated osteoblast differentiation significantly.

REFERENCES

Bellows C G, Aubin J E (1989) Determination of the number ofosteoprogenitors in isolated fetal rat calvarial cells in vitro. DevelopBiol 113:8-13.

Bellows C G, Wang Y H. Heersche J N, Aubin J E (1994)1,25-dihydroxyvitamin D₃ stimulates adipocytic differentiation incultures of fetal rat calvarial cells: comparison with the effects ofdexamethasone. Endocrinology 134:2221-2229.

Friedenstein A J (1976) Precursor cells of mechanocytes. Int Rev Cytol47: 327-355.

Mundy R G (1995) Osteoblests, bone formation and mineralization. In:Bone remodeling and its disorders. Martin Dunitz Ltd pp. 29-30.

Owen M. Friendenstein A J (1988) Stromal stem cells: Marrow-derivedosteogenic precursors. Ciba Found Symp 136:42-60.

Qu Q, Perälä-Heape M, Kapanen A, Dahllund J, Salo J, Väänänen, H K,Härkönen. P (1998) Estrogen enhances differentiation of osteoblasts inmouse bone marrow culture. Bone 22:201-209.

Qu Q, Härkönen P L, Väänänen H K (1999) Comparative effects of estrogenand antiestrogens on differentiation of osteoblasts in mouse bone marrowculture. J Cell Biochem 73: 500-507.

EXAMPLE 4 In vitro Bone formation Assay

The activity of mature osteoblasts can be determined by quantifyingtheir ability to form mineralized bone matrix. This is done bydemineralizing the formed bone matrix, and determining the amount ofcalcium released. Thus, this culture system fulfills requirements of anin vitro model useful for studying the bone formation activity of matureosteoblasts.

Procedure:

The mature osteoblasts obtained during the 8-day secondary culture inthe absence of estrogen and any test substances described above wereallowed to form bone nodules by culturing them for 7 additional days. Atthe end of the culture, the amount of calcium deposited during theculture period was determined, and the amount of bone formation (calciumdeposition) calculated.

In order to quantify the amount of calcium deposited, the cell cultureswere washed three times with Ca²⁺- and Mg²⁺-free PBS and incubatedovernight at room temperature in 0.6M HCl. Extracts of 50 μl werecomplexed with 1 ml determined o-cresol-phthalein-complexon. Thecolorimetric reaction was determined at 570 nm in a spectrophotometer.Absolute calcium concentrations were determined by comparison with acalibrated standard provided by the vendor.

In this study, the effect of lanthanum carbonate on bone formation wastested. The following sample groups were included:

-   -   Baseline (including vehicle)    -   Control (Baseline+10⁻¹⁰ M 17β-estradiol)    -   Baseline+100 ng/ml lanthanum    -   Baseline+500 ng/ml lanthanum    -   Baseline+1000 ng/ml lanthanum    -   Baseline+5000 ng/ml lanthanum    -   Baseline+15000 ng/ml lanthanum        Tables of Results:

The amount of bone formation was determined by measuring the amount ofcalcium deposited into bone nodules formed by mature osteoblasts. Theresults are shown as the amount of calcium released (mmol/L) from thebone nodules after HCl extraction. The baseline values are too low toshow the results using relative amounts as was done in the other assays.TABLE 12 Calcium deposition (mmol/L) in the preliminary bone formationassay Group 1 2 3 4 Mean ± SD Baseline 0 0 0 0 0.00 ± 0.00 Control 0.040 0 0.04 0.02 ± 0.02 LA 100 0 0 0 0 0.00 ± 0.00 LA 500 0 0 0 0.09 0.02 ±0.05 LA 1000 0.10 0 0.11 0.05 0.07 ± 0.05(*) LA 5000 0.59 1.64 0.39 1.621.06 ± 0.66(***) LA 15000 1.48 0.16 0.50 1.41 0.89 ± 0.66(***)

TABLE 13 Calcium deposition (mmol/L) in the first bone formation assayGroup 1 2 3 4 5 6 Mean ± SD Baseline 0 0 0 0.02 0.02 0 0.01 ± 0.01Control 0.15 0.21 0.14 0.10 0.15 0.16 0.15 ± 0.04(***) LA 100 0.04 0.170.01 0.27 0 0.14 0.11 ± 0.11(*) LA 500 0.44 0.15 1.32 0.27 1.31 1.100.77 ± 0.54(***) LA 1000 0.95 1.66 1.47 1.41 1.00 1.25 1.29 ± 0.28(***)LA 5000 1.31 1.55 1.56 1.52 1.40 1.39 1.46 ± 0.10(***) LA 15000 1.461.42 1.56 1.11 1.11 1.08 1.29 ± 0.21(***)

TABLE 14 Calcium deposition (mmol/L) in the second bone formation assayGroup 1 2 3 4 5 6 7 8 Mean + SD Baseline 0 0.01 0 0.01 0 0 0.02 0 0.01 ±0.01 Control 0.22 0.14 0.16 0 0.16 0 0.10 0.16 0.12 ± 0.08(**) LA 1000.04 0.18 0 0 0 0.28 0.14 0 0.08 ± 0.11 LA 500 0.17 0.30 1.41 0 0.020.46 1.17 1.40 0.62 ± 0.61(*) LA 1000 1.09 0.81 1.34 1.56 1.76 0.02 1.521.02 1.14 ± 0.55(***) LA 5000 1.70 1.44 1.64 1.52 1.08 1.63 1.30 1.481.47 ± 0.20(***) LA 15000 1.24 1.46 1.22 1.68 1.62 1.18 1.21 1.56 1.40 ±0.21(***)

The data shown on tables 13 and 14 were combined and analyzed. Theresults from table 12 were not included as there was no significantdifference between the baseline and the control groups. The combinedresults are shown on table 15 and FIG. 4. TABLE 15 Combined results ofthe effects of LA 100-LA 15000 on bone formation activity of matureosteoblasts Group number Mean ± SD Baseline 14 0.01 ± 0.01 Control 140.13 ± 0.06(***) LA 100 14 0.09 ± 0.10(**) LA 500 14 0.68 ± 0.56(***) LA1000 14 1.20 ± 0.45(***) LA 5000 14 1.47 ± 0.16(***) LA 15,000 14 1.35 ±0.21(***)Results:

All concentrations of the lanthanum (III) ion tested showed a highlysignificant activation of the bone formation activity of matureosteoblasts, the activation being highest with the highest testconcentrations. The control substance, 17β-estradiol, activated boneformation significantly.

REFERENCES

Bellows C G, Aubin J E (1989) Determination of the number ofosteoprogenitors in isolated fetal rat calvarial cells in vitro. DevBiol 113:8-13.

Bellows C G, Wang Y H, Heersche J N, Aubin J E (1994)1,25-dihydroxyvitamin D₃ stimulates adipocytic differentiation incultures of fetal rat calvarial cells: comparison with the effects ofdexamethasone. Endocrinology 134:2221-2229.

Friedenstein A J (1976) Precursor cells of mechanocytes. Int Rev Cytol47:327-355.

Mundy R G (1995) Osteoblasts, bone formation and mineralization. In:Bone remodeling and its disorders. Martin Dunitz Ltd pp. 29-30.

Owen M, Friendentein A J (1988) Stromal stem cells: Marrow-derivedosteogenic precursors. Ciba Found Symp 136:42-60.

Qu Q, Perälä-Heape M, Kapanen A, Dahllund J, Salo J, Väänänen H K,Härkönen, P (1998) Estrogen enhances differentiation of osteoblasts inmouse bone marrow culture. Bone 22:201-209.

Qa Q, Härkönen P L, Väänänen H K (1999) Comparative effects of oestrogenand antiestrogens on differentiation of osteoblasts in mouse bone marrowculture. J Cell Biochem 73: 500-507.

Animals for in Vitro Studies Species/strain/age/sex Supplier Mouse/NMRI<8-12 w, male and University of Turku, The centre of female experimentalanimals, Turku, Finland Rat, Sprague-Dawley, 1 day University of Turku,The centre of experimental animals, Turku, FinlandStatistical Analyses of in Vitro Results

The mean and standard deviation (SD) of each group was determined.One-way analysis of variance (ANOVA) was used to study if the valuesobtained between different groups (baseline vs. controls and testsubstances) were statistically different (with p<0.05). Statisticalsignificance is shown in each table and figure with asterisks, oneasterisk (*) indicating a p-value between 0.05 and 0.01, two asterisks(**) a p-value between 0.01 and 0.001, and three asterisks (***) ap-value<0.001. No asterisks indicate that the results of the group donot differ significantly from the results of the corresponding baselinegroup.

Summary of in Vitro Results

The effects of the test concentrations of the lanthanum (III) ion on theactivity and differentiation of bone cells are summarized on table 17,where (+) means significant activation, (−) significant inhibition, and(0) no effect. One character (+ or −) means a p-value between 0.05 and0.01, two characters (++ or −−) a p-value between 0.01 and 0.001, andthree characters (+++ or −−−) a p-value<0.001. TABLE 17 The effects ofLA on bone cells Dose, Bone Osteoclast Osteoblast Bone ng/ml resorptiondifferentiation differentiation formation 100 0 −− + ++ 500 0 0 0 +++1000 0 −− 0 +++ 5000 0 −−− −−− +++ 15000 0 −−− −−− +++Conclusions of in Vitro Studies

The lanthanum (III) ion is a powerful stimulator of the bone formationactivity of mature osteoblasts at all concentrations tested, the bestresponses observed with the highest test concentrations (LA 5000 and LA15000). However, these concentrations may also have cytotoxic effects onthe osteoblast precursor cells, which may compensate the activation ofmature osteoblasts in vivo.

LA 500 and LA 1000 also stimulate bone formation, but theseconcentrations do not decrease the formulation of osteoblasts in theosteoblast differentiation assay, suggesting that they have no cytotoxiceffects on osteoblast precursor cells. However, LA 1000 decreases theformation of osteoclasts in osteoclast differentiation assay, suggestingthat it may have cytotoxic effects on osteoclast precursor cells. Theonly significant effect of LA 500 in the four assays was the activationof bone formation. Thus, this concentration of LA may be useful inincreasing the bone formation without cytotoxic effects.

LA 100 appears to activate both bone formation and osteoblastdifferentiation, and inhibit osteoclast differentiation and boneresorption (although the inhibition of bone resorption is notstatistically significant). All these effects would strengthen bones.

EXAMPLE 5 In Vivo Bone Formation Study

Procedure:

The specimens taken from the iliac crest of growing immature dogs wereanalysed. The group was divided into a control and treatment group. Thetreatment group received 1000 mg/Kg of lanthanum carbonate administeredorally twice daily. The groups were run for 13 weeks, after which timesamples of bone were taken vertically through the iliac crest, embeddedin methyl methacrylate based resin, sectioned and stained with toluidineblue and Von Kossa stain. The parameters measured were:

-   -   Trabecular and cortical bone mass    -   Osteoid surface and volume    -   Osteoblast surface    -   Cortical osteoid volume    -   Trabecular and cortical osteoclast number    -   Resorptive surfaces in cortex and trabecular bone        Results:

The iliac crest of these animals is acting as a growth plate. Theappearances are those of immature animals actively growing. There wasvery active bone remodeling throughout the specimens sampled and, inaddition, there appeared to be bone modelling with very activeperiosteal osteoclasis on the cortical surface, and within the cortex onthe other.

There was a marked difference in cortical thickness between thedifferent animals and marked variation in the amount of bone within thebiopsy specimen. This degree of variation was not restricted to eitherof the two groups of animals, or to animals of particular sex.

There was a statistically significant difference for the trabecular bonevolume between the two groups. The trabecular bone volume was lower inthe control group (approximately half that in the treatment group) thanin the lanthanum treated group. There was no statistically significantdifference in any of the other bone parameters investigated between thetwo groups.

There was an increase of trabecular bone volume in treated animals(about twice) compared to the control group. These results suggest thatlanthanum influences bone growth at the growth plate.

For the purposes of Examples 6 and 7, the term “hydrated lanthanumcarbonate” refers to lanthanum carbonate having a water contentapproximately equivalent to 4-5 moles of water.

EXAMPLE 6 Preparation of Stabilized Hydrated Lanthanum Carbonate andVitamin D Chewable Tablets (250 mg, 500 mg, 750 mg, and 1000 mg)

The manufacturing process involves sieving and blending the activeingredients with the excipients followed by direct compression. Morespecifically the steps are as follows:

a) Blend the lanthanum carbonate, the vitamin D and the excipients(e.g., dextrates, colloidal silicon dioxide, talc (optional) andmagnesium stearate).

b) Compress the blend using standard tooling to the target compressionweight.

The following formulations illustrate tablets that can be made using theabove manufacturing technique. TABLE 18A Formulation A 250 mg Ingredienttablet 500 mg tablet Function Active Ingredients: Hydrated lanthanum477.0 mg 954.0 mg Active (III) carbonate Vitamin D₂ Tablet 0.47 mg 0.94mg Active Grade (850,000 IU/gram) Other Ingredients: Dextrates 1246.53mg 2493.06 mg Stabilizes lanthanum carbonate Colloidal anhydrous 36.0 mg72.0 mg Improves blending silica and flow Purified talc 30.0 mg 60.0 mgLubricant or glidant Magnesium stearate 10.0 mg 20.0 mg Lubricant Total1800 mg 3600 mg

TABLE 18B Formulation B 250 mg Ingredient tablet 500 mg tablet FunctionActive Ingredients: Hydrated lanthanum 477.0 mg 954.0 mg Active (III)carbonate Vitamin D₃ Tablet 0.47 mg 0.94 mg Active Grade (850,000IU/gram) Other Ingredients: Dextrates 1246.53 mg 2493.06 mg Stabilizeslanthanum carbonate Colloidal anhydrous 36.0 mg 72.0 mg Improvesblending silica and flow Purified talc 30.0 mg 60.0 mg Lubricant orglidant Magnesium stearate 10.0 mg 20.0 mg Lubricant Total 1800 mg 3600mg

TABLE 18C Formulation C 250 mg Ingredient tablet 500 mg tablet FunctionActive Ingredients: Hydrated lanthanum 477.0 mg 954.0 mg Active (III)carbonate Vitamin D₃ Tablet 4.0 mg 8.0 mg Active Grade (100,000 IU/gram)Other Ingredients: Dextrates 1243 mg 2486 mg Stabilizes lanthanumcarbonate Colloidal anhydrous 36.0 mg 72.0 mg Improves blending silicaand flow Purified talc 30.0 mg 60.0 mg Lubricant or glidant Magnesiumstearate 10.0 mg 20.0 mg Lubricant Total 1800 mg 3600 mg

TABLE 18D Formulation D 250 mg tablet 500 mg tablet 750 mg tablet 1000mg tablet Tablet 13 mm 18 mm 20 mm 22 mm diameter Formulation Lanthanum250 mg 500 mg 750 mg 1000 mg carbonate as elemental lanthanum Hydrated477 mg 954 mg 1431 mg 1908 mg lanthanum carbonate Vitamin D₂ 0.47 mg0.47 mg 0.94 mg 0.94 mg Tablet Grade 850,000 IU/gram Dextrates(hydrated) 532.7 mg 1065.9 mg 1598.7 mg 2131.9 mg Colloidal 21.2 mg 42.4mg 63.6 mg 84.8 mg silicon dioxide Magnesium 10.6 mg 21.2 mg 31.8 mg42.4 mg stearate Total weight 1042 mg 2048 mg 3126 mg 4168 mg

TABLE 18E Formulation E Percent by Weight Formulation Components in theTablet Hydrated lanthanum carbonate 45.8% Vitamin D₃ Tablet Grade(100,000 IU/gram) 0.4% Colloidal silicon dioxide (e.g., Aerosil ® 2.1%200 available from Degussa Corp. (Piscataway, NJ)) Dextrates 50.7%Magnesium sterate 1.0%

TABLE 18F Formulation F Percent by Weight Formulation Components in theTablet Hydrated lanthanum carbonate 63.6% Vitamin D₃ Tablet Grade(100,000 IU/gram) 0.5% Glyceryl dibehenate 3.0% Colloidal silicondioxide (e.g., Aerosil ® 200) 2.0% Sorbitol 29.9% Talc 1.0%

TABLE 18G Formulation G Percent by Weight Formulation Components in theTablet Hydrated lanthanum carbonate 63.6% Vitamin D₃ Tablet Grade(100,000 IU/gram) 0.5% Glyceryl dibehenate 3.0% Colloidal silicondioxide (e.g., Aerosil ® 200) 2.0% Mannitol 29.9% Talc 1.0%

TABLE 18H Formulation H Percent by Weight Formulation Components in theTablet Hydrated lanthanum carbonate 63.6% Vitamin D₃ Tablet Grade(100,000 IU/gram) 0.5 Glyceryl dibehenate 3.0% Colloidal silicon dioxide(e.g., Aerosil ® 200) 2.0% Xylitol 29.9% Talc 1.0%

EXAMPLE 7 Preparation of Soft Gelatin Capsule Formulations of HydratedLanthanum Carbonate and 25-hydroxy Vitamin D₂ or 1,25-dihydroxy VitaminD₂

The manufacture of soft gelatin capsule formulations of hydratedlanthanum carbonate and 25-hydroxy vitamin D₂ or 1,25-dihydroxy vitaminD₂ includes the following steps:

(1) 25-hydroxy vitamin D₂ or 1,25-dihydroxy vitamin D₂ and, optionally,an antioxidant (such as BHA (i.e., butylated hydroxyanisole availablefrom Eastman Chemical Company, Kingsport, Tenn.), BHT (i.e., butylatedhydroxytoluene available from Eastman Chemical Company, Kingsport,Tenn.) or DL-alpha-tocopherol (available from BASF, Florham Park, N.J.)are dissolved in a medium chain triglyceride (e.g., Miglyol™ 812available from Sasol, Houston, Tex.);

(2) hydrated lanthanum carbonate is added as a suspension to form apaste; and

(3) the paste (i.e., fill composition having a maximum volume of 1 mL)would be encapsulated in a shell consisting of gelatin, glycerol, water,and, optionally, a plasticizer such as sorbitol (available fromRoquette, Lestrem, France).

Below are examples of shell and fill compositions:

Shell composition #1 for a soft gelatin device: said compositioncomprising 38.0-46.0% by weight of gelatin, 14-25% by weight of sorbitolsolution 70% (non crystallizable), 0.2-0.6% by weight of glycine,0.02-0.03% by weight of butylated hydroxy anisole and 40.5-45.5% byweight of purified water.

Shell composition #2 for a soft gelatin device: said compositioncomprising 38.0-46.0% by weight of gelatin, 14-25% by weight of sorbitolsolution, 70% (non crystallizable), 0.2-0.6% by weight of glycine,0.02-0.03% by weight of butylated hydroxy anisole, 0.02-0.03% by weightof butylated hydroxy toluene, 40.5-45.5% by weight of Purified water.Fill Composition #1 Lanthanum Carbonate hydrated 477.0 mg 25-hydroxyvitamin D₂ 0-1.0 mg Butylated Hydroxyanisole 0.2 mg Medium ChainTriglyceride (e.g., MIGLYOL ™ 812) to 1000 mg Fill Composition #2Lanthanum Carbonate hydrated 477.0 mg 25-hydroxy vitamin D₂ 0-1.0 mgButylated Hydroxytoluene 0.2 mg Medium Chain Triglyceride (e.g.,MIGLYOL ™ 812) to 1000 mg Fill Composition #3 Lanthanum Carbonatehydrated 477.0 mg 25-hydroxy vitamin D₂ 0-1.0 mg D-alpha tocopherol 20mg Medium Chain Triglyceride (e.g., MIGLYOL ™ 812) to 1000 mg FillComposition #4 Lanthanum Carbonate hydrated 477.0 mg 1,25-dihydroxyvitamin D₂ 0-1.0 mg Butylated Hydroxyanisole 0.2 mg Medium ChainTriglyceride (e.g., MIGLYOL ™ 812) to 1000 mg Fill Composition #5Lanthanum Carbonate hydrated 477.0 mg 1,25-dihydroxy vitamin D₂ 0-1.0 mgButylated Hydroxytoluene 0.2 mg Medium Chain Triglyceride (e.g.,MIGLYOL ™ 812) to 1000 mg Fill Composition #6 Lanthanum Carbonatehydrated 477.0 mg 1,25-dihydroxy vitamin D₂ 0-1.0 mg D-alpha tocopherol20 mg Medium Chain Triglyceride (e.g., MIGLYOL ™ 812) to 1000 mg

Having illustrated and described the principals of the invention inpreferred embodiments, it should be appreciated to those skilled in theart that the invention can be modified in arrangement and detail withoutdeparture from such principals. We claim all modifications coming withthe scope of the following claims.

All publications, patents and patent applications referred to herein areincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

1. A pharmaceutical composition for the treatment or prevention of abone deficit condition or bone remodeling disorder comprising alanthanum (III) compound and a bone enhancing agent.
 2. A compositionaccording to claim 1, wherein the lanthanum (III) compound is alanthanum salt, a lanthanum chelate, a lanthanum resin, a lanthanumabsorbent, or a mixture thereof.
 3. A composition according to claim 2,wherein the lanthanum (III) compound is the lanthanum salt, wherein thelanthanum salt is a lanthanum carbonate, a lanthanum carbonate hydrate,a lanthanum chloride, or a mixture thereof.
 4. A composition accordingto claim 3, wherein the lanthanum (III) salt is the lanthanum carbonatehydrate.
 5. A composition according to claim 1, wherein the boneenhancing agent is a synthetic hormone, a natural hormone, estrogen,calcitonin, tamoxifen, a bisphosphonate, a bisphosphonate analog,vitamin D, a vitamin D analog, a mineral supplement, a statin drug, aselective estrogen receptor modulator, a sodium fluoride, or a mixturethereof.
 6. A composition according to claim 5, wherein the boneenhancing agent is vitamin D.
 7. A composition according to claim 1,wherein the lanthanum (III) compound is lanthanum carbonate tetrahydrateand the bone enhancing agent is vitamin D.
 8. A composition according toclaim 1, wherein the lanthanum (III) compound is present in thecomposition in an amount from about 125 mg to about 1000 mg as elementallanthanum.
 9. A composition according to claim 6, wherein the boneenhancing agent is 25-hydroxy vitamin D₂ and is present in thecomposition in an amount of up to about 1 mg.
 10. A compositionaccording to claim 6, wherein the bone enhancing agent is 1,25-dihydroxyvitamin D₂ and is present in the composition in an amount of up to about1 mg.
 11. A pharmaceutical dosage for the treatment or prevention of abone deficit condition or bone remodeling disorder comprising alanthanum (III) compound and a bone enhancing agent, wherein thelanthanum (III) compound and the bone enhancing agent are in separatedosage forms.
 12. The pharmaceutical dosage of claim 11, wherein thebone enhancing agent is vitamin D.